DESCRIPTION: DRADA is a nuclear enzyme that converts multiple adenosines to isosines in double-helical RNA substrates without apparent intrinsic sequence specificity. In spite of its conservation throughout the animal kingdom, the in vivo role of DRADA has remained obscure since its discovery in 1987. However, our recent findings and studies by others strongly suggest that DRADA acts as a nuclear RNA editing enzyme to regulate the expression of important gene transcripts such as the glutamate-gated ion channel receptors (GluR) of the central nervous system. During the last period of grant support, the applicant succeeded in isolating cDNA clones for DRADA. Using these clones to express recombinant DRADA protein, she and her colleagues have obtained preliminary evidence that demonstrate an involvement of DRADA in GluR RNA editing. In this application, they propose to address questions focussing on the biological functions and natural substrate RNAs of DRADA. Using an established in vitro assay system, they will investigate the mechanism of GluR-B RNA editing in depth, as a model gene system to better understand DRADA action. The interaction of DRADA and GluR-B RNA with an essential co-factor needed for site-selctive editing will be investigated. It is proposed that the co-factor be purified, and its cDNA cloned. The purified co-factor protein will be used to reconstitute the GluR-B RNA editing with DRADA in vitro. The applicant will also determine directly the repertoire of in vivo target RNAs of DRADA. The information gained by the proposed experiments should lead to enhanced understanding of the full range of biological functions of this intriguing nuclear enzyme.